The long-term objective of the research program is to induce immune tolerance to allogeneic organ transplants such that immunosuppressive drugs are not required to maintain permanent graft acceptance. Previous studies in the program have shown that tolerance to heart allografts can be achieved using combined heart and bone marrow transplantation in wild-type MHC-mismatched murine hosts that have been conditioned with total lymphoid irradiation (TLI) and anti-thymocyte serum (ATS). The hosts become stable mixed chimeras without the development of graft versus host disease (GVHD). Tolerance induction and prevention of GVHD is dependent on host regulatory natural killer (NK) T cells that become the predominant residual T cell subset after TLI and ATS conditioning. Our recent studies show that tolerance and GVHD prevention is also dependent on the development of donor Treg cells and host Treg cells that are not NK T cells. A hypothesis that explains the results is that host NK T cells interact with host and donor APC's, and then augment/activate the non-NK Treg cells that provide alloantigen specificity for tolerance induction. Whereas the regulatory activity of the NK T cells is IL-4 dependent, that of the non-NK Treg cells is IL-4 and IL-10 dependent. The hypothesis will be tested by adding back purified NK T cells and non-NK Treg cells from wild-type, Treg deficient, and cytokine deficient (i.e. IL-4"'", IL-10"'") host and donor type mice to appropriate TLI/ATS conditioned hosts. The conditioned hosts will receive combined MHC-mismatched organ and bone marrow transplants, and graft acceptance and GVHD will be monitored. The phenotype and cytokine dependence of the non-NK Treg cells will be determined as well as the dependence of NK T cell activation on interaction with APC's. Our recent studies show that the NK T cells in wild type mice are far more resistant to apoptosis induced by TLI/ATS conditioning than conventional T cells. However, the differential resistance is lost in p53"'" mice and in Bcl-2 transgenic mice. We will determine whether the ability to induce tolerance and prevent GVHD is also lost in the latter genetically altered mice.